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Emer S Ferro - One of the best experts on this subject based on the ideXlab platform.
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Intracellular peptides: From discovery to function
Eupa Open Proteomics, 2014Co-Authors: Emer S Ferro, Vanessa Rioli, Leandro M Castro, Lloyd D. FrickerAbstract:Abstract Peptidomics techniques have identified hundreds of peptides that are derived from proteins present mainly in the cytosol, mitochondria, and/or nucleus; these are termed intracellular peptides to distinguish them from secretory pathway peptides that function primarily outside of the cell. The proteasome and Thimet Oligopeptidase participate in the production and metabolism of intracellular peptides. Many of the intracellular peptides are common among mouse tissues and human cell lines analyzed and likely to perform a variety of functions within cells. Demonstrated functions include the modulation of signal transduction, mitochondrial stress, and development; additional functions will likely be found for intracellular peptides.
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AGH is a new hemoglobin alpha-chain fragment with antinociceptive activity
Peptides, 2013Co-Authors: Natalia Ribeiro, Emer S Ferro, Vanessa Rioli, Lilian C Russo, Leandro M Castro, Elaine F. Toniolo, Camila S. DaleAbstract:Abstract Limited proteolysis of certain proteins leads to the release of endogenous bioactive peptides. Hemoglobin-derived peptides such as hemorphins and hemopressins are examples of intracellular protein-derived peptides that have antinociceptive effects by modulating G-protein coupled receptors activities. In the present study, a previously characterized substrate capture assay that uses a catalytically inactive form of the Thimet Oligopeptidase was combined with isotopic labeling and mass spectrometry in order to identify new bioactive peptides. Indeed, we have identified the peptide AGHLDDLPGALSAL (AGH), a fragment of the hemoglobin alpha-chain, which specifically bind to the inactive Thimet Oligopeptidase in the substrate capture assay. Previous peptidomics studies have identified the AGH as well as many other natural peptides derived from hemoglobin alpha-chain containing this sequence, further suggesting that AGH is a natural endogenous peptide. Pharmacological assays suggest that AGH inhibits peripheral inflammatory hyperalgesic responses through indirect activation of mu opioid receptors, without having a central nervous system activity. Therefore, we have successfully used the substrate capture assay to identify a new endogenous bioactive peptide derived from hemoglobin alpha-chain.
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inhibition of Thimet Oligopeptidase by sirna alters specific intracellular peptides and potentiates isoproterenol signal transduction
FEBS Letters, 2012Co-Authors: Lilian C Russo, Leandro M Castro, Fabio C Gozzo, Emer S FerroAbstract:Mammalian cells have a large number of intracellular peptides that are generated by extralysosomal proteases. In this study, the enzymatic activity of Thimet Oligopeptidase (EP24.15) was inhibited in human embryonic kidney (HEK) 293 cells using a specific siRNA sequence. The semi-quantitative intracellular peptidome analyses of siRNA-transfected HEK293 cells shows that the levels of specific intracellular peptides are either increased or decreased upon EP24.15 inhibition. Decreased expression of EP24.15 was sufficient to potentiate luciferase gene reporter activation by isoproterenol (1–10 μM). The protein kinase A inhibitor KT5720 (1 μM) reduced the positive effect of the EP24.15 siRNA on isoproterenol signaling. Thus, EP24.15 inhibition by siRNA modulates the levels of specific intracellular peptides and isoproterenol signal transduction.
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natural intracellular peptides can modulate the interactions of mouse brain proteins and Thimet Oligopeptidase with 14 3 3e and calmodulin
Proteomics, 2012Co-Authors: Lilian C Russo, Antonio C M Camargo, Vanessa Rioli, Leandro M Castro, Fabio C Gozzo, Amanda F Asega, Henning Ulrich, Priscilla D Negraes, Lilian Cruz, Emer S FerroAbstract:Protein interactions are crucial for most cellular process. Thus, rationally designed peptides that act as competitive assembly inhibitors of protein interactions by mimicking specific, determined structural elements have been extensively used in clinical and basic research. Recently, mammalian cells have been shown to contain a large number of intracellular peptides of unknown function. Here, we investigate the role of several of these natural intracellular peptides as putative modulators of protein interactions that are related to Ca2+-calmodulin (CaM) and 14-3-3e, which are proteins that are related to the spatial organization of signal transduction within cells. At concentrations of 1–50 μM, most of the peptides that are investigated in this study modulate the interactions of CaM and 14-3-3e with proteins from the mouse brain cytoplasm or recombinant Thimet Oligopeptidase (EP24.15) in vitro, as measured by surface plasmon resonance. One of these peptides (VFDVELL; VFD-7) increases the cytosolic Ca2+ concentration in a dose-dependent manner but only if introduced into HEK293 cells, which suggests a wide biological function of this peptide. Therefore, it is exciting to suggest that natural intracellular peptides are novel modulators of protein interactions and have biological functions within cells.
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the cysteine rich protein Thimet Oligopeptidase as a model of the structural requirements for s glutathiolation and oxidative oligomerization
PLOS ONE, 2012Co-Authors: Alberto Malvezzi, Emer S Ferro, Leandro M Castro, Fabio C Gozzo, Patricia M Higa, Antonia Tavares Do Amaral, Gustavo M Silva, Leandro Fornias Machado De Rezende, Gisele MonteiroAbstract:Thimet Oligopeptidase (EP24.15) is a cysteine-rich metallopeptidase containing fifteen Cys residues and no intra-protein disulfide bonds. Previous work on this enzyme revealed that the oxidative oligomerization of EP24.15 is triggered by S-glutathiolation at physiological GSSG levels (10–50 µM) via a mechanism based on thiol-disulfide exchange. In the present work, our aim was to identify EP24.15 Cys residues that are prone to S-glutathiolation and to determine which structural features in the cysteinyl bulk are responsible for the formation of mixed disulfides through the reaction with GSSG and, in this particular case, the Cys residues within EP24.15 that favor either S-glutathiolation or inter-protein thiol-disulfide exchange. These studies were conducted by in silico structural analyses and simulations as well as site-specific mutation. S-glutathiolation was determined by mass spectrometric analyses and western blotting with anti-glutathione antibody. The results indicated that the stabilization of a thiolate sulfhydryl and the solvent accessibility of the cysteines are necessary for S-thiolation. The Solvent Access Surface analysis of the Cys residues prone to glutathione modification showed that the S-glutathiolated Cys residues are located inside pockets where the sulfur atom comes into contact with the solvent and that the positively charged amino acids are directed toward these Cys residues. The simulation of a covalent glutathione docking onto the same Cys residues allowed for perfect glutathione posing. A mutation of the Arg residue 263 that forms a saline bridge to the Cys residue 175 significantly decreased the overall S-glutathiolation and oligomerization of EP24.15. The present results show for the first time the structural requirements for protein S-glutathiolation by GSSG and are consistent with our previous hypothesis that EP24.15 oligomerization is dependent on the electron transfer from specific protonated Cys residues of one molecule to previously S-glutathionylated Cys residues of another one.
Antonio C M Camargo - One of the best experts on this subject based on the ideXlab platform.
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natural intracellular peptides can modulate the interactions of mouse brain proteins and Thimet Oligopeptidase with 14 3 3e and calmodulin
Proteomics, 2012Co-Authors: Lilian C Russo, Antonio C M Camargo, Vanessa Rioli, Leandro M Castro, Fabio C Gozzo, Amanda F Asega, Henning Ulrich, Priscilla D Negraes, Lilian Cruz, Emer S FerroAbstract:Protein interactions are crucial for most cellular process. Thus, rationally designed peptides that act as competitive assembly inhibitors of protein interactions by mimicking specific, determined structural elements have been extensively used in clinical and basic research. Recently, mammalian cells have been shown to contain a large number of intracellular peptides of unknown function. Here, we investigate the role of several of these natural intracellular peptides as putative modulators of protein interactions that are related to Ca2+-calmodulin (CaM) and 14-3-3e, which are proteins that are related to the spatial organization of signal transduction within cells. At concentrations of 1–50 μM, most of the peptides that are investigated in this study modulate the interactions of CaM and 14-3-3e with proteins from the mouse brain cytoplasm or recombinant Thimet Oligopeptidase (EP24.15) in vitro, as measured by surface plasmon resonance. One of these peptides (VFDVELL; VFD-7) increases the cytosolic Ca2+ concentration in a dose-dependent manner but only if introduced into HEK293 cells, which suggests a wide biological function of this peptide. Therefore, it is exciting to suggest that natural intracellular peptides are novel modulators of protein interactions and have biological functions within cells.
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interaction with calmodulin is important for the secretion of Thimet Oligopeptidase following stimulation
FEBS Journal, 2009Co-Authors: Lilian C Russo, Antonio C M Camargo, Marc J Glucksman, Leandro M Castro, Camila N Goni, Amanda F Asega, Cleber A Trujillo, Henning Ulrich, Cristoforo Scavone, Emer S FerroAbstract:Thimet Oligopeptidase (EC 3.4.24.15; EP24.15) was originally described as a neuropeptide-metabolizing enzyme, highly expressed in the brain, kidneys and neuroendocrine tissue. EP24.15 lacks a typical signal peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. In this study, we identified a novel calcium-dependent interaction between EP24.15 and calmodulin, which is important for the stimulated, but not constitutive, secretion of EP24.15. We demonstrated that, in vitro, EP24.15 and calmodulin physically interact only in the presence of Ca2+, with an estimated Kd value of 0.52 μm. Confocal microscopy confirmed that EP24.15 colocalizes with calmodulin in the cytosol of resting HEK293 cells. This colocalization markedly increases when cells are treated with either the calcium ionophore A23187 or the protein kinase A activator forskolin. Overexpression of calmodulin in HEK293 cells is sufficient to greatly increase the A23187-stimulated secretion of EP24.15, which can be inhibited by the calmodulin inhibitor calmidazolium. The specific inhibition of protein kinase A with KT5720 reduces the A23187-stimulated secretion of EP24.15 and inhibits the synergistic effects of forskolin with A23187. Treatment with calmidazolium and KT5720 nearly abolishes the stimulatory effects of A23187 on EP24.15 secretion. Together, these data suggest that the interaction between EP24.15 and calmodulin is regulated within cells and is important for the stimulated secretion of EP24.15 from HEK293 cells. Structured digital abstract • MINT-7148420: EP24.15 (uniprotkb:P52888) and Calmodulin (uniprotkb:P62161) bind (MI:0407) by surface plasmon resonance (MI:0107) • MINT-7148437: EP24.15 (uniprotkb:P52888) and Calmodulin (uniprotkb:P62158) colocalize (MI:0403) by surface plasmon resonance (MI:0107) • MINT-7148406: Calmodulin (uniprotkb:P62161) binds (MI:0407) to EP24.15 (uniprotkb:P52888) by pull down (MI:0096)
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a comparative conformational analysis of Thimet Oligopeptidase ec 3 4 24 15 substrates
Journal of Peptide Research, 2009Co-Authors: S G Jacchieri, Luiz Juliano, M Gomes, Antonio C M CamargoAbstract:The specificity of Thimet Oligopeptidase (EC 3.4.24.15) (TOP 24.15) does not agree with theoretical models devised to explain the specificity characteristic of peptidases toward certain sequences of amino acid residues. According to previous studies peptide chains hydrolyzed by TOP 24.15 adopt similar main chain conformations, although with different and in some cases small probabilities of occurrence in aqueous solution. To determine specific structural features recognized by TOP 24.15, a conformational search including eight polypeptides with known susceptibilities for catalytic hydrolysis was executed and the distribution of each main chain conformation found in the search was tabulated. Two sets of main chain conformations were selected, those common to all peptides in the study and those common only to substrates of TOP 24.15. The former set is very small and includes mainly extended conformations. In contrast, the latter set is large and its conformations are coiled and exhibit sharp turns coincident with positions of hydrolysis by TOP 24.15. These results indicate a possible basis for the selectivity of TOP 24.15.
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a structure based site directed mutagenesis study on the neurolysin ec 3 4 24 16 and Thimet Oligopeptidase ec 3 4 24 15 catalysis
FEBS Letters, 2003Co-Authors: Vitor Oliveira, Antonio C M Camargo, Luiz Juliano, Vanessa Rioli, Maria A Juliano, Mauricio De Campos Araujo, Ivarne L S Tersariol, Emer S FerroAbstract:Abstract Neurolysin (EP24.16) and Thimet Oligopeptidase (EP24.15) are closely related metalloendopeptidases. Site-directed mutagenesis of Tyr613 (EP24.16) or Tyr612 (EP24.15) to either Phe or Ala promoted a strong reduction of kcat/KM for both enzymes. These data suggest the importance of both hydroxyl group and aromatic ring at this specific position during substrate hydrolysis by these peptidases. Furthermore, the EP24.15 A607G mutant showed a kcat/KM of 2×105 M−1 s−1 for the Abz-GFSIFRQ-EDDnp substrate, similar to that of EP24.16 (kcat/KM=3×105 M−1 s−1) which contains Gly at the corresponding position; the wild type EP24.15 has a kcat/KM of 2.5×104 M−1 s−1 for this substrate.
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temperature and salts effects on the peptidase activities of the recombinant metalloOligopeptidases neurolysin and Thimet Oligopeptidase
FEBS Journal, 2002Co-Authors: Vitor Oliveira, Antonio C M Camargo, Emer S Ferro, Vanessa Rioli, Maria A Juliano, Reynaldo M Gatti, Alberto Spisni, Luiz JulianoAbstract:We report the recombinant neurolysin and Thimet Oligopeptidase (TOP) hydrolytic activities towards internally quenched fluorescent peptides derived from the peptide Abz-GGFLRRXQ-EDDnp (Abz, ortho-aminobenzoicacid; EDDnp, N-(2,4-dinitrophenyl) ethylenediamine), in which X was substituted by 11 different natural amino acids. Neurolysin hydrolyzed these peptides at R–R or at R–X bonds, and TOP hydrolyzed at R–R or L–R bonds, showing a preference to cleave at three or four amino acids from the C-terminal end. The kinetic parameters of hydrolysis and the variations of the cleavage sites were evaluated under different conditions of temperature and salt concentration. The relative amount of cleavage varied with the nature of the substitution at the X position as well as with temperature and NaCl concentration. TOP was activated by all assayed salts in the range 0.05–0.2 m for NaCl, KCl, NH4Cl and NaI, and 0.025–0.1 m for Na2SO4. Concentration higher than 0.2 N NH4Cl andNaI reduced TOP activity, while 0.5 N or higher concentration of NaCl, KCl and Na2SO4 increased TOP activity. Neurolysin was strongly activated by NaCl, KCl and Na2SO4, while NH4Cl and NaI have very modest effect. High positive values of enthalpy (ΔH*) and entropy (ΔS*) of activation were found together with an unusual temperature dependence upon the hydrolysis of the substrates. The effects of low temperature and high NaCl concentration on the hydrolytic activities of neurolysin and TOP do not seem to be a consequence of large secondary structure variation of the proteins, as indicated by the far-UV CD spectra. However, the modulation of the activities of the two Oligopeptidases could be related to variations of conformation, in limited regions of the peptidases, enough to modify their activities.
Luiz Juliano - One of the best experts on this subject based on the ideXlab platform.
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catalytic properties of Thimet Oligopeptidase h600a mutant
Biochemical and Biophysical Research Communications, 2010Co-Authors: Mauricio F M Machado, Emer S Ferro, Luiz Juliano, Marcelo F Marcondes, Vanessa Rioli, Maria A Juliano, Vitor OliveiraAbstract:Thimet Oligopeptidase (EC 3.4.24.15, TOP) is a metallo-Oligopeptidase that participates in the intracellular metabolism of peptides. Predictions based on structurally analogous peptidases (Dcp and ACE-2) show that TOP can present a hinge-bend movement during substrate hydrolysis, what brings some residues closer to the substrate. One of these residues that in TOP crystallographic structure are far from the catalytic residues, but, moves toward the substrate considering this possible structural reorganization is His{sup 600}. In the present work, the role of His{sup 600} of TOP was investigated by site-directed mutagenesis. TOP H600A mutant was characterized through analysis of S{sub 1} and S{sub 1}' specificity, pH-activity profile and inhibition by JA-2. Results showed that TOP His{sup 600} residue makes important interactions with the substrate, supporting the prediction that His{sup 600} moves toward the substrate due to a hinge movement similar to the Dcp and ACE-2. Furthermore, the mutation H600A affected both K{sub m} and k{sub cat}, showing the importance of His{sup 600} for both substrate binding and/or product release from active site. Changes in the pH-profile may indicate also the participation of His{sup 600} in TOP catalysis, transferring a proton to the newly generated NH{sub 2}-terminus or helping Tyr{sup 605} and/or Tyr{sup 612}more » in the intermediate oxyanion stabilization.« less
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a comparative conformational analysis of Thimet Oligopeptidase ec 3 4 24 15 substrates
Journal of Peptide Research, 2009Co-Authors: S G Jacchieri, Luiz Juliano, M Gomes, Antonio C M CamargoAbstract:The specificity of Thimet Oligopeptidase (EC 3.4.24.15) (TOP 24.15) does not agree with theoretical models devised to explain the specificity characteristic of peptidases toward certain sequences of amino acid residues. According to previous studies peptide chains hydrolyzed by TOP 24.15 adopt similar main chain conformations, although with different and in some cases small probabilities of occurrence in aqueous solution. To determine specific structural features recognized by TOP 24.15, a conformational search including eight polypeptides with known susceptibilities for catalytic hydrolysis was executed and the distribution of each main chain conformation found in the search was tabulated. Two sets of main chain conformations were selected, those common to all peptides in the study and those common only to substrates of TOP 24.15. The former set is very small and includes mainly extended conformations. In contrast, the latter set is large and its conformations are coiled and exhibit sharp turns coincident with positions of hydrolysis by TOP 24.15. These results indicate a possible basis for the selectivity of TOP 24.15.
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characterization of Thimet Oligopeptidase and neurolysin activities in b16f10 nex2 tumor cells and their involvement in angiogenesis and tumor growth
Molecular Cancer, 2007Co-Authors: Thaysa Paschoalin, Luiz Juliano, Maria A Juliano, Vitor Oliveira, Adriana K Carmona, Elaine G Rodrigues, Hugo P Monteiro, Luiz R TravassosAbstract:Background: Angiogenesis is a fundamental process that allows tumor growth by providing nutrients and oxygen to the tumor cells. Beyond the oxygen diffusion limit from a capillary blood vessel, tumor cells become apoptotic. Angiogenesis results from a balance of pro- and antiangiogenic stimuli. Endogenous inhibitors regulate enzyme activities that promote angiogenesis. Tumor cells may express pro-angiogenic factors and hydrolytic enzymes but also kinin-degrading Oligopeptidases which have been investigated. Results: Angiogenesis induced by B16F10-Nex2 melanoma cells was studied in a co-culture with HUVEC on Matrigel. A stimulating effect on angiogenesis was observed in the presence of B16F10Nex2 lysate and plasma membrane. In contrast, the B16F10-Nex2 culture supernatant inhibited angiogenesis in a dose-dependent manner. This effect was abolished by the endo-Oligopeptidase inhibitor, JA-2. Thimet Oligopeptidase (TOP) and neurolysin activities were then investigated in B16F10-Nex2 melanoma cells aiming at gene sequencing, enzyme distribution and activity, influence on tumor development, substrate specificity, hydrolytic products and susceptibility to inhibitors. Fluorescence resonance energy transfer (FRET) peptides as well as neurotensin and bradykinin were used as substrates. The hydrolytic activities in B16F10-Nex2 culture supernatant were totally inhibited by o-phenanthrolin, JA-2 and partially by Pro-Ile. Leupeptin, PMSF, E-64, Z-Pro-Prolinal and captopril failed to inhibit these hydrolytic activities. Genes encoding M3A enzymes in melanoma cells were cloned and sequenced being highly similar to mouse genes. A decreased proliferation of B16F10-Nex2 cells was observed in vitro with specific inhibitors of these Oligopeptidases. Active rTOP but not the inactive protein inhibited melanoma cell development in vivo increasing significantly the survival of mice challenged with the tumor cells. On Matrigel, rTOP inhibited the bradykinin – induced angiogenesis. A possible regulation of the homologous tumor
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the role of tyr605 and ala607 of Thimet Oligopeptidase and tyr606 and gly608 of neurolysin in substrate hydrolysis and inhibitor binding
Biochemical Journal, 2007Co-Authors: Mauricio F M Machado, Emer S Ferro, Luiz Juliano, Vanessa Rioli, Maria A Juliano, Leandro M Castro, Fernanda M Dalio, Ivarne L S Tersariol, Vitor OliveiraAbstract:The physicochemical properties of TOP (Thimet Oligopeptidase) and NEL (neurolysin) and their hydrolytic activities towards the FRET (fluorescence resonance energy transfer) peptide series Abz-GFSXFRQ-EDDnp [where Abz is o-aminobenzoyl; X=Ala, Ile, Leu, Phe, Tyr, Trp, Ser, Gln, Glu, His, Arg or Pro; and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] were compared with those of site-mutated analogues. Mutations at Tyr605 and Ala607 in TOP and at Tyr606 and Gly608 in NEL did not affect the overall folding of the two peptidases, as indicated by their thermal stability, CD analysis and the pH-dependence of the intrinsic fluorescence of the protein. The kinetic parameters for the hydrolysis of substrates with systematic variations at position P1 showed that Tyr605 and Tyr606 of TOP and NEL respectively, played a role in subsite S1. Ala607 of TOP and Gly608 of NEL contributed to the flexibility of the loops formed by residues 600–612 (GHLAGGYDGQYYG; one-letter amino acid codes used) in NEL and 599–611 (GHLAGGYDAQYYG; one-letter amino acid codes used) in TOP contributing to the distinct substrate specificities, particularly with an isoleucine residue at P1. TOP Y605A was inhibited less efficiently by JA-2 {N-[1-(R,S)-carboxy-3-phenylpropyl]Ala-Aib-Tyr-p-aminobenzoate}, which suggested that the aromatic ring of Tyr605 was an important anchor for its interaction with wild-type TOP. The hydroxy groups of Tyr605 and Tyr606 did not contribute to the pH-activity profiles, since the pKs obtained in the assays of mutants TOP Y605F and NEL Y606F were similar to those of wild-type peptidases. However, the pH–kcat/Km dependence curve of TOP Y605A differed from that of wild-type TOP and from TOP Y606F. These results provide insights into the residues involved in the substrate specificities of TOP and NEL and how they select cytosolic peptides for hydrolysis.
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a structure based site directed mutagenesis study on the neurolysin ec 3 4 24 16 and Thimet Oligopeptidase ec 3 4 24 15 catalysis
FEBS Letters, 2003Co-Authors: Vitor Oliveira, Antonio C M Camargo, Luiz Juliano, Vanessa Rioli, Maria A Juliano, Mauricio De Campos Araujo, Ivarne L S Tersariol, Emer S FerroAbstract:Abstract Neurolysin (EP24.16) and Thimet Oligopeptidase (EP24.15) are closely related metalloendopeptidases. Site-directed mutagenesis of Tyr613 (EP24.16) or Tyr612 (EP24.15) to either Phe or Ala promoted a strong reduction of kcat/KM for both enzymes. These data suggest the importance of both hydroxyl group and aromatic ring at this specific position during substrate hydrolysis by these peptidases. Furthermore, the EP24.15 A607G mutant showed a kcat/KM of 2×105 M−1 s−1 for the Abz-GFSIFRQ-EDDnp substrate, similar to that of EP24.16 (kcat/KM=3×105 M−1 s−1) which contains Gly at the corresponding position; the wild type EP24.15 has a kcat/KM of 2.5×104 M−1 s−1 for this substrate.
Alan J Barrett - One of the best experts on this subject based on the ideXlab platform.
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85 Thimet Oligopeptidase
Handbook of Proteolytic Enzymes (Second Edition)#R##N#Aspartic and Metallo Peptidases, 2004Co-Authors: Alan J BarrettAbstract:Publisher Summary This chapter elaborates the activity, specificity and structural chemistry of Thimet Oligopeptidase (TOP). Human TOP cleaved hexa-alanine, but not tetra- or penta-alanine. TOP is an Oligopeptidase with a sharply-defined upper limit of substrate size. Heptade-capeptide substrates include γ-endorphin and nociceptin/orphanin FQ. In a study of the specificity of TOP, it is confirmed that all substrates contain 17 or fewer amino acids. The rules governing the substrate specificity of TOP remain unclear, but with both substrates and inhibitors, hydrophobic residues tend to be favored in positions PI and P3′, and Pro in P2′. The enzyme also shows a preference for cleaving bond three to six residues from the C-terminus. TOP is a single-chain protein of about 78.5 kDa that does not contain intramolecular disulfide bonds. There are no indications of post-translational modifications in the biosynthesis of TOP or of a proteolytically activatable proenzyme. Directed mutagenesis has been used to identify amino acid residues important for the catalytic activity of TOP.
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85 – Thimet Oligopeptidase
Handbook of Proteolytic Enzymes, 2004Co-Authors: Alan J BarrettAbstract:Publisher Summary This chapter elaborates the activity, specificity and structural chemistry of Thimet Oligopeptidase (TOP). Human TOP cleaved hexa-alanine, but not tetra- or penta-alanine. TOP is an Oligopeptidase with a sharply-defined upper limit of substrate size. Heptade-capeptide substrates include γ-endorphin and nociceptin/orphanin FQ. In a study of the specificity of TOP, it is confirmed that all substrates contain 17 or fewer amino acids. The rules governing the substrate specificity of TOP remain unclear, but with both substrates and inhibitors, hydrophobic residues tend to be favored in positions PI and P3′, and Pro in P2′. The enzyme also shows a preference for cleaving bond three to six residues from the C-terminus. TOP is a single-chain protein of about 78.5 kDa that does not contain intramolecular disulfide bonds. There are no indications of post-translational modifications in the biosynthesis of TOP or of a proteolytically activatable proenzyme. Directed mutagenesis has been used to identify amino acid residues important for the catalytic activity of TOP.
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Thimet Oligopeptidase site directed mutagenesis disproves previous assumptions about the nature of the catalytic site
FEBS Letters, 1998Co-Authors: Jinqmay Chen, Richard A E Stevens, Paul W Wray, Neil D Rawlings, Alan J BarrettAbstract:Abstract Zinc metallopeptidases that contain the His-Glu-Xaa-Xaa-His (HEXXH) motif generally have a third ligand of the metal ion that may be either a Glu residue (in clan MA) or a His residue (in clan MB) (Rawlings and Barrett (1995) Methods Enzymol. 248, 183–228). Thimet Oligopeptidase has not yet been assigned to either clan, and both Glu and His residues have been proposed as the third ligand. We mutated candidate ligand residues in the recombinant enzyme and identified Glu, His and Asp residues that are important for catalytic activity and/or stability of the protein. However, neither of the Glu and His residues close to the HEXXH motif that have previously been suggested to be ligands is required for the binding of zinc. We conclude that Thimet Oligopeptidase is not a member of clan MA or clan MB and it is likely that the enzyme possesses a catalytic site and protein fold different from those identified in any metallopeptidase to date. The definitive identification of the third zinc ligand may well require the determination of the crystallographic structure of Thimet Oligopeptidase or one of its homologues.
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rat Thimet Oligopeptidase large scale expression in escherichia coli and characterization of the recombinant enzyme
Biochemical Journal, 1995Co-Authors: N Mckie, Pamela M Dando, M A Brown, Alan J BarrettAbstract:The coding sequence for rat testis Thimet Oligopeptidase (TOP) (EC 3.4.24.15) was placed under the control of the T7 polymerase/promoter system. Cultures of Escherichia coli transfected with the resulting plasmid expressed the enzyme as a soluble cytoplasmic protein. Medium-scale cultures allowed isolation of the enzyme in quantities of tens of milligrams. The availability of the recombinant enzyme permitted the determination of such chemical properties as epsilon 280 (48,960), zinc content (2 atom/molecule) and available thiol content (8-10/molecule) for TOP. The recombinant enzyme showed the catalytic activities previously reported for the naturally occurring enzyme, so that we can now conclude with confidence that these are all due to TOP and there is no need to postulate the existence of separate ‘Pz-peptidase’ or ‘endo-Oligopeptidase A’ enzymes.
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Thimet Oligopeptidase specificity evidence of preferential cleavage near the c terminus and product inhibition from kinetic analysis of peptide hydrolysis
Biochemical Journal, 1995Co-Authors: C G Knight, Pamela M Dando, Alan J BarrettAbstract:The substrate-size specificity of human Thimet Oligopeptidase (EC 3.4.24.15) was investigated with oligomers of glycyl-prolyl-leucine (GPL)n where n = 2, 3, 4 and 5. These peptides were cleaved only at Leu-Gly bonds to give GPL as the single final product. Hydrolysis was most rapid with (GPL)3 and slowest with (GPL)5. The more water-soluble oligomers of Gly-Hyp-Leu showed the same trend. (Gly-Hyp-Leu)6 was not hydrolysed, consistent with the previous finding that substrates larger than 17 amino acids are not cleaved by Thimet Oligopeptidase. The cleavage of (GPL)3 to GPL fitted a sequential first-order model. First-order kinetics were unexpected as the initial substrate concentration was greater than Km. The anomaly was also seen during the cleavage of bradykinin and neurotensin, and in these cases first-order behaviour was due to potent competitive inhibition by the C-terminal product. The sequential mechanism for (GPL)3 breakdown by Thimet Oligopeptidase does not discriminate between initial cleavages towards the N- or C-terminus. As isoleucine is an unfavourable residue in P1, substrates were made in which selected leucine residues were replaced by isoleucine. GPL--GPI--GPL (where--represents the bond between the tripeptide units) was resistant to hydrolysis and GPI--GPL--GPL was cleaved only at the -Leu-Gly- bond. Experiments with isoleucine-containing analogues of (Gly-Hyp-Leu)4 showed that Thimet Oligopeptidase preferred to cleave these peptides near the C-terminus.
Marc J Glucksman - One of the best experts on this subject based on the ideXlab platform.
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EP24.15 as a Potential Regulator of Kisspeptin Within the Neuroendocrine Hypothalamus
Endocrinology, 2015Co-Authors: Nicole Woitowich, Keith D. Philibert, Randy J. Leitermann, Manida Wungjiranirun, Janice H. Urban, Marc J GlucksmanAbstract:The neuropeptide kisspeptin (Kiss1) is integral to the advent of puberty and the generation of cyclical LH surges. Although many complex actions of Kiss1 are known, the mechanisms governing the processing/regulation of this peptide have not been unveiled. The metallo enzyme, endopeptidase 24.15 (Thimet Oligopeptidase), has been demonstrated to play a key role in the processing and thus the duration of action of the reproductive neuropeptide, GnRH, which signals downstream of Kiss1. Initial in silico modeling implied that Kiss1 could also be a putative substrate for EP24.15. Coincubation of Kiss1 and EP24.15 demonstrated multiple cleavages of the peptide predominantly between Arg29-Gly30 and Ser47-Phe48 (corresponding to Ser5-Phe6 in Kiss-10; Kiss-10 as a substrate had an additional cleavage between Phe6-Gly7) as determined by mass spectrometry. Vmax for the reaction was 2.37±0.09 pmol/min · ng with a Km of 19.68 ± 2.53μM, which is comparable with other known substrates of EP24.15. EP24.15 immunoreactivity...
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interaction with calmodulin is important for the secretion of Thimet Oligopeptidase following stimulation
FEBS Journal, 2009Co-Authors: Lilian C Russo, Antonio C M Camargo, Marc J Glucksman, Leandro M Castro, Camila N Goni, Amanda F Asega, Cleber A Trujillo, Henning Ulrich, Cristoforo Scavone, Emer S FerroAbstract:Thimet Oligopeptidase (EC 3.4.24.15; EP24.15) was originally described as a neuropeptide-metabolizing enzyme, highly expressed in the brain, kidneys and neuroendocrine tissue. EP24.15 lacks a typical signal peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. In this study, we identified a novel calcium-dependent interaction between EP24.15 and calmodulin, which is important for the stimulated, but not constitutive, secretion of EP24.15. We demonstrated that, in vitro, EP24.15 and calmodulin physically interact only in the presence of Ca2+, with an estimated Kd value of 0.52 μm. Confocal microscopy confirmed that EP24.15 colocalizes with calmodulin in the cytosol of resting HEK293 cells. This colocalization markedly increases when cells are treated with either the calcium ionophore A23187 or the protein kinase A activator forskolin. Overexpression of calmodulin in HEK293 cells is sufficient to greatly increase the A23187-stimulated secretion of EP24.15, which can be inhibited by the calmodulin inhibitor calmidazolium. The specific inhibition of protein kinase A with KT5720 reduces the A23187-stimulated secretion of EP24.15 and inhibits the synergistic effects of forskolin with A23187. Treatment with calmidazolium and KT5720 nearly abolishes the stimulatory effects of A23187 on EP24.15 secretion. Together, these data suggest that the interaction between EP24.15 and calmodulin is regulated within cells and is important for the stimulated secretion of EP24.15 from HEK293 cells. Structured digital abstract • MINT-7148420: EP24.15 (uniprotkb:P52888) and Calmodulin (uniprotkb:P62161) bind (MI:0407) by surface plasmon resonance (MI:0107) • MINT-7148437: EP24.15 (uniprotkb:P52888) and Calmodulin (uniprotkb:P62158) colocalize (MI:0403) by surface plasmon resonance (MI:0107) • MINT-7148406: Calmodulin (uniprotkb:P62161) binds (MI:0407) to EP24.15 (uniprotkb:P52888) by pull down (MI:0096)
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hydrogen bond residue positioning in the 599 611 loop of Thimet Oligopeptidase is required for substrate selection
FEBS Journal, 2008Co-Authors: Lisa A Bruce, Marc J Glucksman, Jeffrey A Sigman, Danica Randall, Scott Rodriguez, Michelle M Song, Donald E Elmore, Amanda Pabon, Adele J WolfsonAbstract:Thimet Oligopeptidase (EC 3.4.24.15) is a zinc(II) endopeptidase implicated in the processing of numerous physiological peptides. Although its role in selecting and processing peptides is not fully understood, it is believed that flexible loop regions lining the substrate-binding site allow the enzyme to conform to substrates of varying structure. This study describes mutant forms of Thimet Oligopeptidase in which Gly or Tyr residues in the 599–611 loop region were replaced, individually and in combination, to elucidate the mechanism of substrate selection by this enzyme. Decreases in kcat observed on mutation of Tyr605 and Tyr612 demonstrate that these residues contribute to the efficient cleavage of most substrates. Modeling studies showing that a hinge-bend movement brings both Tyr612 and Tyr605 within hydrogen bond distance of the cleaved peptide bond supports this role. Thus, molecular modeling studies support a key role in transition state stabilization of this enzyme by Tyr605. Interestingly, kinetic parameters show that a bradykinin derivative is processed distinctly from the other substrates tested, suggesting that an alternative catalytic mechanism may be employed for this particular substrate. The data demonstrate that neither Tyr605 nor Tyr612 is necessary for the hydrolysis of this substrate. Relative to other substrates, the bradykinin derivative is also unaffected by Gly mutations in the loop. This distinction suggests that the role of Gly residues in the loop is to properly orientate these Tyr residues in order to accommodate varying substrate structures. This also opens up the possibility that certain substrates may be cleaved by an open form of the enzyme.
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Hydrogen bond residue positioning in the 599–611 loop of Thimet Oligopeptidase is required for substrate selection
FEBS Journal, 2008Co-Authors: Lisa A Bruce, Marc J Glucksman, Jeffrey A Sigman, Danica Randall, Scott Rodriguez, Michelle M Song, Donald E Elmore, Amanda Pabon, Adele J WolfsonAbstract:Thimet Oligopeptidase (EC 3.4.24.15) is a zinc(II) endopeptidase implicated in the processing of numerous physiological peptides. Although its role in selecting and processing peptides is not fully understood, it is believed that flexible loop regions lining the substrate-binding site allow the enzyme to conform to substrates of varying structure. This study describes mutant forms of Thimet Oligopeptidase in which Gly or Tyr residues in the 599–611 loop region were replaced, individually and in combination, to elucidate the mechanism of substrate selection by this enzyme. Decreases in kcat observed on mutation of Tyr605 and Tyr612 demonstrate that these residues contribute to the efficient cleavage of most substrates. Modeling studies showing that a hinge-bend movement brings both Tyr612 and Tyr605 within hydrogen bond distance of the cleaved peptide bond supports this role. Thus, molecular modeling studies support a key role in transition state stabilization of this enzyme by Tyr605. Interestingly, kinetic parameters show that a bradykinin derivative is processed distinctly from the other substrates tested, suggesting that an alternative catalytic mechanism may be employed for this particular substrate. The data demonstrate that neither Tyr605 nor Tyr612 is necessary for the hydrolysis of this substrate. Relative to other substrates, the bradykinin derivative is also unaffected by Gly mutations in the loop. This distinction suggests that the role of Gly residues in the loop is to properly orientate these Tyr residues in order to accommodate varying substrate structures. This also opens up the possibility that certain substrates may be cleaved by an open form of the enzyme.
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flexibility in substrate recognition by Thimet Oligopeptidase as revealed by denaturation studies
Biochemical Journal, 2005Co-Authors: Jeffrey A Sigman, Marc J Glucksman, Tasneem H Patwa, Ana V Tablante, Calleen D Joseph, Adele J WolfsonAbstract:Thimet Oligopeptidase (TOP) is a soluble metalloendopeptidase belonging to a family of enzymes including neurolysin and neprilysin that utilize the HEXXH metal-binding motif. TOP is widely distributed among cell types and is able to cleave a number of structurally unrelated peptides. A recent focus of interest has been on structure–function relationships in substrate selectivity by TOP. The enzyme’s structural fold comprises two domains that are linked at the bottom of a deep substrate-binding cleft via several flexible loop structures. In the present study, fluorescence spectroscopy has been used to probe structural changes in TOP induced by the chemical denaturant urea. Fluorescence emission, anisotropy and collisional quenching data support a two-step unfolding process for the enzyme in which complete loss of the tertiary structure occurs in the second step. Complete loss of activity and loss of catalytic Zn(II) from the active site, monitored by absorption changes of the metal chelator 4-(2-pyridylazo)resorcinol, are also connected with the second step. In contrast, the first unfolding event, which is linked to changes in the noncatalytic domain, leads to a sharp increase in kcat towards a 9-residue substrate and a sharp decrease in kcat for a 5-residue substrate. Thus a conformational change in TOP has been directly correlated with a change in substrate selectivity. These results provide insight into how the enzyme can process the range of structurally unrelated peptides necessary for its many physiological roles.
